PROJECT SUMMARY/ABSTRACT Glioblastoma is a devastating primary brain tumor, which is characterized by rapid growth and resistance to conventional therapies. The disease is rare with an estimated prevalence of less than 25,000 cases in the United States. The overall median survival is approximately 15 months after treatment with standard therapies, including surgery, radiotherapy, and chemotherapy. Cancer recurrence following initial therapy is common and especially devastating, as treatment options are limited and mostly ineffective for prolonging survival. The median survival time for patients with recurrent glioblastoma is 3 to 7 months. Thus, new treatment approaches are critically needed for patients in this setting. Oncolytic virotherapy represents an attractive novel therapeutic approach for recurrent glioblastoma. In this application, a potent oncolytic virus, DNX-2401, will be delivered to human brain tumors and evaluated in a human Phase II clinical study to assess clinical efficacy. DNX-2401 is a conditionally replicative oncolytic virus that functions by replicating in, and killing cells lacking growth control caused by any defect in the retinoblastoma (Rb) pathway, which is defective in 90% of glioblastomas. DNX-2401 also appears to trigger an antitumor immune response that can lead to tumor killing and increased survival in some patients. Results from Phase I clinical studies indicate that DNX-2401 can: (1) replicate in human glioblastoma tumors for a period of weeks to months, (2) elicit tumor necrosis within weeks of injection, (3) trigger intratumoral immune cell infiltration, and (4) lead to long term tumor destruction detectable by MRI scans. In 2014, DNX-2401 was granted orphan drug designation for the treatment of glioma in the US and EU and Fast Track Status for recurrent glioblastoma in the US. This open-label, multicenter, Phase II clinical study will determine the efficacy of DNX-2401 when administered as a monotherapy to patients with recurrent glioblastoma or gliosarcoma. Up to 112 patients will be enrolled and receive a single DNX-2401 dose of 3 X 1010 viral particles injected directly into the tumor at the time of a tumor biopsy procedure. Enrolled patients will then be followed for 18 months to assess efficacy and safety, and every 4 months thereafter for survival. The specific aims of the study are to: 1) prospectively determine the objective response rate (ORR) in recurrent glioblastoma, 2) to determine the rate of 12 month overall survival, the clinical benefit rate, and changes in quality of life, and 3) to evaluate the rate and severity of adverse events after treatment with DNX-2401.